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. 2009 Mar;28(3):103–108. doi: 10.1089/dna.2008.0792

FIG. 3.

FIG. 3.

Expression and purification of EnvC from 143B cells infected with rVV–GFP–envC. (A–B) Infected cells were solubilized in Laemmli buffer and fractionated in gradient PAGE gel (4–12%); proteins were transferred to the PVDF membrane and probed with (A) anti-HIV-1 human IgG isolated from serum of an HIV-1–infected patient and (B) normal human IgG. (C–D) Analysis of purified mutant HIV envelope glycoproteins produced in 143B cells infected with rVV–GFP–envC. The mutant HIV EnvC yielded the expected band of approximately 140 kDa under denaturing conditions. (C) SDS-PAGE Coomassie blue staining. Lane 1, 10 μg of total protein from mock-infected cells; lane 2, 10 μg of protein purified by lentil lectin affinity column and Superdex-S200 size exclusion column chromatography using extracts of cells infected with rVV–GFP–envC. (D) Silver staining. Lane 1, total protein (2 μg) after the lentil lectin affinity column purification; lane 2, purified protein (2 μg) after size exclusion column chromatography purification.